Proteins and polypeptides are naturally occuring, and recently, synthetically prepared compounds that are compound of long chains of amino acids. Proteins are found throughout living things and function as enzymes, hormones, immunoglobulins, structural elements, and other constituents of living things. Research regarding the structure and function of a protein often requires that the amino acid sequence (primary structure) of the protein be determined in order for a protein or the polypeptide constituents thereof to be synthesized, the sequence of amino acids must be determined. In the search involving the function of proteins, the primary structure must first be determined in an attempt to elucidate the mechanism of action of the protein. In recombinant DNA methodology, the primary structure must be determined to elucidate the corresponding structure of a DNA or RNA encoding the same.
The sequence of amino acids in proteins or polypeptides is commonly determined by stepwise chemical degradation in which single amino acids are derivatized and removed one by one from the end of the polypeptide to be identified. The standard method, the Edman degradation (Edman, P., Acta Chem. Scand., (1950) 4, 283), is the preferred method. Alternatively, thioacylation has attracted considerable attention as an effective alternative to the Edman degradation, owing to the particularly mild conditions under which it is employed.
The thioacylation degradation of proteins and polypeptides was first proposed by Barrett (Barrett, G. C., Chem. Comm., (1967) 487) as an alternative to the Edman degradation. The process involves reacting the N-terminal amino acid of a starting polypeptide immobilized on an insoluble support (heterogeneous phase reaction) with a thioacylating reagent in an alkaline aqueous or anhydrous solvent. The excess reagent is removed by washing of the immobilized polypeptide, and byproducts of the reaction resulting from the decomposition of the reagent are similarly removed, to yield the N-thioacyl polypeptide, Formula I, wherein X is an alkyl or aryl substituent and R is representative of the various amino acid side chains. Thioacylating reagents exhibit low solubility in aqueous media which limits their use in homogenous phase techniques. This limitation was overcome by the advent of solid phase and pulsed liquid methods. ##STR1##
Various activated carbodithioic esters, thiono esters and thiono imides including carboxymethyl dithiobenzoate, cyanomethyl dithiobenzoate, m-nitrobenzoylthionocholine, N-thiobenzoylsuccinimide, thioacetylthioglycholic acid and methyl dithoacetate have been employed as reagents for the sequential degradation of polypeptides. Aminolysis of carbodithioic esters is susceptible to general base catalysis and experimental conditions under which simple alkyl esters of aliphatic dithioacids, such as methyl dithioacetate, behave as satisfactory thiocylating reagents have been reported (Previero, A., Gourdol, A., Derancourt, J. and Coletti-Previero, M.-A., FEBS Lett., (1975) 51, 68).
In the second step, the N-thiocayl polypeptide is subjected to cleavage by volatile anhydrous acid to afford the 2-substituted-5(4H)-thiazolinone of the N-terminal amino acid, Formula II, wherein X is an alkyl or aryl substituent and R is representative of the various amino acid side chains. Thioacylation offers a significant advantage over the Edman degradation in that the cleavage reaction is short in duration and occurs under relatively mild conditions. Also liberated during the cleavage reaction is the salt of the residual polypeptide, which is the starting polypeptide with the N-terminal amino acid removed. ##STR2##
2-Methyl-5(4H)-thiazolinones were historically identified by hydrolytic regeneration of the free amino acids (Mross, Jr., G. A., (1971) Ph.D. Dissertation, University of California, San Diego). Consequently, serine, threonine and tryptophan were not recovered in high yield owing to their instability during hydrolysis. Alternatively, 2-methyl-5(4H)-thiazolinones have been identified by gas liquid chromatography, preferably, after reaction with excess acetic anhydride in pyridine or acetyl chloride in TFA (Simpson, D. L., Hranisewljevic, J. and Davidson, E. A., Biochemistry, (1972) 11, 1849) which yields the corresponding 5-O-acetyl-2-metylthiazoles. 2-Phenyl-5(4H)-thiazolinones have been identified directly by mass spectrometry and by thin layer chromatography after conversion to the corresponding N-thiobenzoyl amino acid anilides (Barrett, G. C. and Khokhar, A. R., J. Chromatog., (1969) 39, 47).
In the Edman degradation, the product of the cleavage reaction is the 2-anilino-5(4H)-thiazolinone (ATZ). This unstable species is then converted to its isomer, the 3-phenyl-2-thiohydantoin (PTH), Formula III, wherein R is representative of the various amino acid side chains. ##STR3##
Conventional techniques for conversion involve heating the ATZ (50.degree.-80.degree. C.) under nitrogen in hydrochloric acid, anhydrous methanolic hydrochloric acid or anhydrous methanolic trifluoroacetic acid. Conversion with aqueous acid invariably results in deamidation of asparagine and glutamine to their respective acids, while anhydrous methanolic hydrochloric and trifluoroacetic acids cause esterification of acidic residues. Certain PTH amino acids require adjustment of pH or time for quantitative conversion while other PTH amino acids tend to decompose during the conversion reaction. While alcoholysis of ATZ amino acids has been recognized as an intermediate reaction during conversion in alcoholic acid, the isolation of the intermediate phenylthiocarbamyl amino acid esters is not feasible as a consequence of their tendency toward conversion to the corresponding PTH amino acids (Tarr, G. E., in Methods of Protein Microcharacterization, Shively, J. E., Ed., Humana Press, Clifton, N.J. (1986) 175-179).